Determining an Amount of Analyte in Plasma Based on a Measurement of an Amount of Analyte in a Whole-Blood Sample

The present invention relates to various aspects of determining an amount of analyte in plasma based on a measurement of an amount of analyte in a whole-blood sample.

Analyzer units for measuring amounts of analytes in blood samples by means of respective detectors or sensors are widely used in the medical and clinical field. Such analyzer units are often simply referred to as analyzers.

In addition to general requirements in respect of accuracy, precision, and reliability, analyzer units for clinical applications are often subject to further critical constraints. Such constraints include a need for low operating costs, low down times, ease of use, efficiency of use, in particular reduced need for sample preparation, etc.

Many analyzer units perform measurements on biological samples that comprise blood with all its components, in particular including the plasma as well as the red blood cells. This has the advantage that the need for pre-processing the blood sample prior to performing the desired measurements is kept to a minimum, if not eliminated altogether. Generally, a blood sample including both plasma and red blood cells is referred to as a “whole blood” sample.

Usually, the concentration of at least some analytes is defined as the concentration of the analyte in plasma. A direct measurement of the concentration of the analyte in plasma based on a whole-blood sample would thus initially require the separation of the whole-blood sample into plasma and other blood components, in particular red blood cells. However, this is a time-consuming process.

It is therefore desirable to determine the amount of analyte in plasma directly from a measurement of an amount of the analyte in a whole-blood sample.

To this end, U.S. Pat. No. 10,132,800 proposes a method for measuring an analyte amount in a whole-blood sample, including: measuring the hematocrit level of the whole-blood sample; measuring an analyte amount directly in the whole-blood sample; and calculating a corrected analyte amount according to relation: D=P(D, D) where Dis the corrected analyte amount, Dis the measured analyte amount, Dis the measured hematocrit level, and Pis a non-constant polynomial of a degree greater than or equal to 1 having as indeterminate values the measured analyte amount, D, and the measured hematocrit level, D, and having its polynomial coefficients depending on the analyte.

However, multivariate polynomials having two independent variables require relatively many polynomial coefficients to be determined in a calibration process, thus rendering the process complex and prone to overfitting. For example, even a lowest order multivariate polynomial including only a single cross-term includes four polynomial coefficients that need to be determined by calibration.

Accordingly, it remains desirable to provide a method for measuring an analyte amount in a whole-blood sample that is accurate and robust and that requires only little calibration efforts, or that at least provides an alternative to known methods.

It is further desirable to provide a method that corrects for the deviation of measurements on whole-blood samples from corresponding measurements on plasma samples without the need for repeated calibration of the individual analyzer units by the operator of the analyzer unit. Instead, it is desirable that the applicable correction can be determined during design or manufacturing of the device and/or of the associated assays and that can be sent to each analyzer unit.

On this background, according to a first aspect, disclosed herein are embodiments of a method for calibrating a group of analyzer units, each analyzer unit of the group of analyzer units configured for determining an amount of an analyte in plasma of a whole-blood sample. Embodiments of the method comprise:

a) providing a plurality of calibration whole-blood samples, the plurality of calibration whole-blood samples including calibration whole-blood samples having respective hematocrit levels,

b) for each calibration whole-blood sample of the plurality of calibration whole-blood samples:

c) generating a nonlinear functional relationship between the computed ratios and the corresponding hematocrit measurement values by curve fitting of a nonlinear function parametrized by one or more calibration parameters, the curve fitting resulting in respective parameter values of the one or more calibration parameters;

d) storing a representation of the fitted nonlinear function in each analyzer unit of the group of analyzer units to allow each analyzer unit of the group of analyzer units to compute a hematocrit correction factor.

The representation of the fitted nonlinear function is a representation of the parametrized nonlinear function where the one or more calibration parameters have the parameter values resulting from the curve fitting. Once calibrated, each of the analyzer units of the group of analyzer units may determine an amount of the analyte in plasma of a whole-blood sample by measuring a whole-blood measurement value indicative of an amount of the analyte in a whole-blood sample and a hematocrit measurement value indicative of a hematocrit level of the whole-blood sample, by determining a hematocrit correction factor from the hematocrit measurement value and from the stored representation of the fitted nonlinear function, including the fitted parameter values, and by applying the hematocrit correction factor to the whole-blood measurement value.

In particular, according to one aspect, disclosed herein are embodiments of a method for measuring an amount of analyte in plasma of a whole-blood sample; wherein the method comprises:

Accordingly, the amount of the analyte is computed as a simple product or ratio of the measurement value indicative of the measured amount of analyte in the whole-blood sample and a hematocrit correction factor that depends on the measured hematocrit level. In particular, the hematocrit correction factor is derived from a fitted nonlinear function of an indeterminate variable representing the hematocrit level, the fitted nonlinear function being parametrized by one or more calibration parameters. The parameter values of the one or more calibration parameters in the fitted nonlinear function depend on at least the type of analyte and/or the type of assay used for measuring the analyte.

In some embodiments, the nonlinear function only has a single indeterminate variable, in particular the hematocrit level as the only indeterminate variable, thus facilitating an accurate and robust calibration fit using only a few calibration parameters. In some embodiments, the nonlinear function is further dependent on the measured amount of analyte, but preferably with only three or fewer calibration parameters. Preferably, the calibration function is a non-polynomial function, such as including an exponential function.

In some embodiments, the nonlinear function is parameterized by fewer than four calibration parameters, such as three or two calibration parameters or a single calibration parameter.

The inventors have realized that this process provides an accurate determination of the analyte amount in plasma over a large range of analyte amounts and hematocrit levels, while reducing the risk of undesirable overfitting.

Computing the hematocrit correction factor from the stored representation of the fitted nonlinear function may comprise evaluating the fitted nonlinear function at the measured hematocrit value, either by explicit computation of the function value, by look-up in a look-up table and optional interpolation, or in another suitable manner so as to obtain the function value of the fitted non-linear function at the measured hematocrit value. In particular, in some embodiments, the nonlinear function is represented by a look-up table with the hematocrit value as the key, optionally including an interpolation. Applying the computed hematocrit value to the whole-blood measurement value may comprise multiplying the whole-blood measurement value with the computed hematocrit correction factor or dividing the whole-blood measurement value by the computed hematocrit correction factor. This may depend on whether the ratios between the whole-blood and plasma measurement during calibration have been computed by dividing the whole-blood measurement value by the corresponding plasma measurement value or by dividing the plasma measurement value by the corresponding whole-blood measurement value.

Preferably, the calibration process is performed using a more than one calibration analyzer units in order to accommodate for possible variations between analyzer units of the group of analyzer units. Accordingly, in some embodiments, step b) of the method for calibrating the group of analyzer units comprises, for each of a set of calibration analyzer units of the group of analyzer units, performing the following steps for each of a set of calibration whole-blood samples of the plurality of whole blood samples:

Preferably, the set of calibration analyzer units comprises more than one, such as more than five, such as between 3 and 20, such as between 5 and 10, e.g. between 6 and 10 calibration analyzer units. The calibration analyzer units may be used to perform measurements on the same set of calibration whole-blood sample or on respective sets of calibration whole-blood samples.

In some embodiments, during calibration, the hematocrit measurement value and/or the plasma measurement value are measured by the same calibration analyzer unit used for measuring the corresponding whole-blood measurement value of the calibration whole-blood sample. To this end, a calibration plasma sample may be prepared from each calibration whole-blood sample, e.g. in a known manner, such as by centrifugation. The prepared calibration plasma sample may then be presented to the calibration analyzer unit for measurement of the plasma measurement value indicative of the amount of analyte in the plasma sample obtained from the original whole-blood sample.

Accordingly, in some embodiments, generating the nonlinear functional relationship by curve fitting comprises: generating a plurality of data points, each data point representing a hematocrit measurement value of a calibration whole-blood sample and a corresponding computed ratio between a whole-blood measurement value measured by one of the set of calibration analyzer units on said calibration whole-blood sample and a corresponding plasma measurement value measured by the same one of the set of calibration analyzer units. The corresponding plasma measurement value is preferably measured on a plasma sample obtained from said calibration whole blood sample. The hematocrit measurement value of the calibration whole-blood sample is preferably measured using the same one of the set of calibration analyzer units as the corresponding whole-blood measurement value pertaining to the same data point. Preferably, the curve fitting is based on data points obtained using respective ones of the set of the calibration analyzer units and data points obtained using respective ones of the plurality of calibration whole-blood samples.

In various embodiments, the calibration parameters associated with a particular analyte are determined by curve fitting the parametrized nonlinear function to a generated calibration data set. The calibration data set is derived from measured analyte amounts of said particular analyte in calibration whole-blood samples and in corresponding plasma samples. The measured analyte amounts of said particular analyte in the calibration whole-blood samples are obtained by one or more calibration analyzer units of the same group as the measurement analyzer unit used for the subsequent measurements.

In some embodiments, the parametrized nonlinear function is a nonlinear, non-polynomial function of the hematocrit level. In particular, in some embodiments, the nonlinear, non-polynomial function is an exponential function of the hematocrit level. The inventors have realized that a non-polynomial functions, in particular an exponential function, provides a particular accurate correction factor with a low risk of overfitting, at least for some types of analytes. In some embodiments, the non-polynomial function is a function different from a fraction of two polynomials.

In some embodiments, the hematocrit correction factor HCF (Hct) is calculated from the measured hematocrit level Hct as

where f(Hct) is a function of at least the measured hematocrit level Hct. In some embodiments, the function f is a parametrized function, parametrized by one or more parameters. In some embodiments, the function f has Hct as its only indeterminate while, in other embodiments, the function f is dependent on one or more additional quantities, e.g. temperature and/or the concentration of the analyte to be determined.

In some embodiments, the hematocrit correction factor HCF(Hct) is calculated from the measured hematocrit level Hct as

with calibration parameters a and b. In some embodiments, the calibration parameter a has a parameter value between 2.0 and 2.4, such as between 2.20 and 2.21, such as a=2.204 and wherein the calibration parameter b has a parameter value between 2.2 and 2.7, such as between 2.4 and 2.5, such as between 2.45 and 2.47, such as b=2.468. In another embodiment, the calibration parameter a has a parameter value between 1.9 and 2.0, such as between 1.96 and 1.97 and wherein the calibration parameter b has a parameter value between 1.5 and 1.6, such as between 1.53 and 1.54.

In some embodiments, the hematocrit correction factor HCF (Hct) is calculated from the measured hematocrit level Hct as

with calibration parameters a, b and c, and where conc designates the measured amount of analyte in the whole-blood sample or an approximation thereof. In some embodiments, the calibration parameter a has a parameter value between 0.8 and 3.0, such as between 1.5 and 2.0, such as between 1.7 and 1.9. In some embodiments, the calibration parameter b has a parameter value between 1.5 and 2.0, such as between 1.7 and 1.9. The parameter c may be selected between −0.01 and 1.5, such as between −0.01 and 0.2, or between 0.05 and 1.5. In some embodiments, the parameter c may be selected in dependence of the concentration conc. For example, the parameter c may be determined from a look-up table indexed by the concentration conc. In particular, respective values of c may be associated with different concentration ranges, or the parameter c may be determined by interpolation between parameter values obtained from a look-up table, or otherwise. Accordingly, an accurate calibration may be achieved with relatively few calibration parameters.

In some embodiments, measuring the whole-blood measurement value and/or measuring the plasma measurement value comprises using an immunoassay. The fitted nonlinear function and, hence, the hematocrit correction factor may be specific to the type of immunoassay. The immunoassay may be provided in the form of a replaceable cartridge insertable into the analyzer unit. The fitted nonlinear function and, hence, the hematocrit correction factor may thus be specific to the type of immunoassay but not specific to the particular analyzer unit, as long as the analyzer unit belongs to the group of analyzer unit for which the fitted nonlinear function applies, e.g. all analyzer units of a particular make and model.

In some embodiments the analyte is an antigen. In some embodiments, the analyte is cardiac troponin I. Accordingly, in some embodiments, the whole-blood measurement value is obtained by means of a troponin I assay, in particular a high-sensitivity troponin I assay (hsTnI). Various embodiments of the method disclosed herein provide accurate measurement of analytes, in particular of hsTnI, even when the whole-blood samples are only little diluted. Accordingly, a high sensitivity may be obtained. The inventors have found that a hematocrit correction factor for hsTnI and other analytes, such as NT-proBNP and/or others, can accurately be determined based on hematocrit alone, in particular based on a nonlinear function of only the hematocrit level as indeterminate variable, independently of the analyte concentration. For some analytes, e.g. for Procalcitonin (PCT), while a nonlinear function independently of the analyte concentration may be used, a non-polynomial, nonlinear function that also depends on the analyte concentration may be particularly suitable.

The present disclosure relates to different aspects including the methods described above and in the following, corresponding apparatus, systems, methods, and/or products, each yielding one or more of the benefits and advantages described in connection with one or more of the other aspects, and each having one or more embodiments corresponding to the embodiments described in connection with one or more of the other aspects and/or disclosed in the appended claims.

In particular, according to one aspect, disclosed herein are embodiments of measuring an amount of analyte in plasma of a whole-blood sample using a measurement analyzer unit of a group of analyzer units; wherein the method comprises:

According to another aspect, disclosed herein are embodiments of a computer-implemented method of determining an amount of an analyte in plasma based on a measurement of an amount of the analyte in a whole-blood sample; wherein the method comprises:

Moreover, according to yet another aspect, disclosed herein are embodiments of a data processing system configured to perform the steps of the computer-implemented method described herein. In particular, the data processing system may have stored thereon program code adapted to cause, when executed by the data processing system, the data processing system to perform the steps of the computer-implemented method described herein. The data processing system may be embodied as a single computer or other data processing unit or device, or as a distributed system including multiple computers and/or other data processing devices, e.g. a client-server system, a cloud based system, etc. The data processing system may include a data storage device for storing the computer program and/or sensor data.

In some embodiments, the data processing system is integrated into the analyzer unit, e.g. as a suitably programmed internal data processing unit of the analyzer unit. In other embodiments, the data processing system may be a remote data processing system physically separate from the analyzer unit. To this end, the remote data processing system may include a communications interface for receiving measurement values from the analyzer unit, e.g. via a suitable wired or wireless connection, e.g. directly from the analyzer unit or indirectly via one or more intermediate nodes.

According to one aspect, disclosed herein are embodiments of an analyzer unit for determining an amount of analyte in plasma of a whole-blood sample; wherein the analyzer unit comprises:

The analyzer unit may be an analyzer unit of a group of analyzer unit, all calibrated by the method described herein. In particular all analyzer units of the group of analyzer units may have stored thereon a representation of the same fitted nonlinear function.

The group of analyzer units may comprise or consist of analyzer units of the same make and model or otherwise analyzer units of a selected class of analyzer units that apply the same measurement protocol for measuring the analyte amount and that can therefore apply the same calibration. Hence, the calibration can be performed as part of the design of a particular analyzer unit model and/or during design of a measurement immunoassay to be used by a particular analyzer unit model for measuring a particular type of analyte.

In this respect, it will be appreciated that the parametrized nonlinear function may be analyte-specific and/or specific to a particular type of immunoassay. However, the parametrized nonlinear function may be applicable for a plurality of analyzer units, in particular to a predetermined group of analyzer units such as analyzer units of a particular make and model.

The amount of analyte determined by various embodiments of the process disclosed herein may be an absolute amount or a relative amount, in particular an analyte concentration.

Yet another aspect disclosed herein relates to embodiments of a computer program configured to cause a data processing system to perform the acts of the computer-implemented method described above and in the following. A computer program may comprise program code means adapted to cause a data processing system to perform the acts of the computer-implemented method disclosed above and in the following when the program code means are executed on the data processing system. The computer program may be stored on a computer-readable storage medium, in particular a non-transient storage medium, or embodied as a data signal. The non-transient storage medium may comprise any suitable circuitry or device for storing data, such as a RAM, a ROM, an EPROM, EEPROM, flash memory, magnetic or optical storage device, such as a CD ROM, a DVD, a hard disk, and/or the like.